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OPEN
received: 15 October 2015 accepted: 25 April 2016 Published: 13 May 2016
The P2X4 receptor is required for neuroprotection via ischemic preconditioning Tomohiko Ozaki1,2,3, Rieko Muramatsu1,2,4, Miwa Sasai5, Masahiro Yamamoto5, Yoshiaki Kubota6, Toshiyuki Fujinaka3, Toshiki Yoshimine3 & Toshihide Yamashita1,2 Ischemic preconditioning (IPC), a procedure consisting of transient ischemia and subsequent reperfusion, provides ischemic tolerance against prolonged ischemia in the brain. Although the blood flow changes mediated by IPC are primarily perceived by vascular endothelial cells, the role of these cells in ischemic tolerance has not been fully clarified. In this study, we found that the P2X4 receptor, which is abundantly expressed in vascular endothelial cells, is required for ischemic tolerance following middle artery occlusion (MCAO) in mice. Mechanistically, the P2X4 receptor was stimulated by fluid shear stress, which mimics reperfusion, thus promoting the increased expression of osteopontin, a neuroprotective molecule. Furthermore, we found that the intracerebroventricular administration of osteopontin was sufficient to exert a neuroprotective effect mediated by preconditioning-stimulated P2X4 receptor activation. These results demonstrate a novel mechanism whereby vascular endothelial cells are involved in ischemic tolerance. Ischemic brain injuries resulting from decreases in cerebral blood flow (CBF) are important causes of disability and death. Pharmacological interventions are either ineffective or confounded by adverse effects, and neuroprotection in patients after ischemic brain damage consequently remains a major unfulfilled medical need. Ischemic preconditioning (IPC), defined as transient ischemia and subsequent reperfusion, exerts neuroprotective effects against lethal ischemia. Ischemic tolerance due to preconditioning is observed in experimental animal models. In humans, the number of investigations is currently limited, but ischemic tolerance may naturally occur in human patients1. Therefore, the mechanisms of IPC have attracted considerable attention in terms of their potential applications in adaptive medicine for the treatment of ischemia. Regarding the mechanisms underlying the acquisition of ischemic tolerance, most research has focused on the involvement of neurons and astrocytes. For example, the N-methyl-D-aspartate receptor and the adenosine A1 receptor, both of which are abundantly expressed in neurons2,3, are involved in preconditioning-induced ischemic tolerance4,5. The inhibition of astrocyte-expressed P2X7 receptors attenuates the induction of ischemic tolerance6. In contrast, although IPC is caused by transient blood flow changes1 that are directly perceived by vascular endothelial cells7, the role of these cells in ischemic tolerance has not been clarified. Vascular endothelial cells are exposed to shear stress, in the form of the frictional force exerted by flowing blood. Shear stress mediates the molecular expression in vascular endothelial cells8 and influences vascular phenomena, such as angiogenesis9, atherogenesis10, and related endothelial functions11. These vascular phenomena are well known to partially regulate perivascular cells12; therefore, shear stress-mediated changes in endothelial cell molecular expression may consequently regulate perivascular cell function. Endothelial cell–derived factors, such as endothelin and prostacyclin, reportedly act on perivascular cells, regulating vascular contractility and permeability13,14. Furthermore, vascular endothelial cell–derived molecules can regulate neuronal network 1 Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 2Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. 3Department of Neurosurgery, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 4Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. 5 Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. 6The Laboratory of Vascular Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Correspondence and requests for materials should be addressed to R.M. (email: [email protected]) or T.Y. (email: [email protected])
Scientific Reports | 6:25893 | DOI: 10.1038/srep25893
1
www.nature.com/scientificreports/ formation during both development and repair processes15,16. Based on these findings, we hypothesized that endothelial cell–derived molecules, whose production is increased in response to blood flow changes, such as those involved in IPC, may benefit neurons of the central nervous system (CNS) by providing neuroprotective effects against prolonged ischemia. In this study, we focused on the role of the P2X4 receptor, a subtype of the P2X family of ligand-gated ion channels activated by adenosine triphosphate (ATP). The binding of ATP to the P2X receptor results in the entry of extracellular Ca2+ and activates intracellular signaling that evoke a variety of cellular responses. P2X4 is abundantly expressed in vascular endothelial cells17,18. P2X4−/− mice show weak responses to blood flow changes, such as low Ca2+ influx and subsequent NO production17, indicating that the P2X4 receptor is a mechanoreceptor that senses blood flow-mediated shear stress. Findings on the role of the P2X4 receptor in vascular pathology have been limited to peripheral vascular diseases, such as heart failure19. However, the P2X4 receptor has been detected in the mammalian brain20, but its function in the CNS vasculature remains unknown. Osteopontin, a secreted acidic glycoprotein containing the RGD motif, has multifunctional properties, including adhesive, chemotactic, and cytokine-like properties21. Although osteopontin is expressed in the normal brain21, the level of osteopontin increases under pathological conditions22,23. Therefore, the function of osteopontin has been studied in the context of disease. In terms of ischemic brain injury, osteopontin-deficient mice reportedly show increased neurodegeneration after ischemic cortical stroke24. Moreover, osteopontin administration prevents infarct formation after stroke in mice25. These findings led us to hypothesize that osteopontin is also involved in the endogenous neuroprotective effect mediated by IPC against ischemia, but evidence on this involvement is lacking. Here, we show that the P2X4 receptor, which is known as a shear stress sensor17, is involved in IPC-mediated neuroprotection following middle cerebral artery occlusion (MCAO)-induced cerebral ischemia in mice. P2X4 receptors are expressed by endothelial cells in the adult mouse brain. Treatment with a P2X4 receptor antagonist abolished the ability of IPC to prevent infarct formation and neurological impairment after prolonged ischemia. Shear stress also stimulated P2X4 receptors on vascular endothelial cells, leading to the upregulated expression of osteopontin and a neuroprotective effect25. Thus, neuroprotection by P2X receptor-mediated IPC might involve osteopontin up-regulation.
Results
P2X4 receptor inhibition abolishes IPC–mediated neuroprotection. We first established the experimental design (Fig. 1a) and found that the damage obtained 24 hr after 1 hr of MCAO was prevented when 15 min of MCAO (IPC) was administered 2 day before the prolonged MCAO procedure. We then examined the involvement of the P2X4 receptor in the prevention of infarct formation by IPC before prolonged MCAO. Twenty-four hours after 1 hr of MCAO, mice that received an intracerebroventricular injection of 5-BDBD, a P2X4 receptor antagonist, showed larger infarctions in both the striatum and the cortex than controls (Fig. 1b–d). IPC prevents the progression of neurological deficits caused by MCAO, and we consequently also assessed the involvement of the P2X4 receptor in this process. Mice that received a combination of 5-BDBD pretreatment and IPC showed severe neurological deficits compared with those that did not receive 5BDBD treatment (Fig. 1e). We observed that single-IPC did not form an infarct in the brain (Fig. 1b–d) or cause neurological impairment (Fig. 1e). In addition, 5-BDBD treatment did not exacerbate the infarct formation (Fig. 1b–d) or neurological impairment (Fig. 1e) caused by MCAO. Staining with 2,3,5-triphenyltetrazolium chloride (TTC) remained abundant in the brain and no neurological impairment in the intact mice (Fig. 1b–e). These data indicate that P2X4 receptor inhibition abolished IPC–mediated neuroprotection and the prevention of neurological impairment after MCAO. P2X4 receptor in vascular endothelial cells is required for neuroprotection against prolonged MCAO. To investigate the cell type(s) that are involved in P2X4 receptor-mediated neuroprotec-
tion, we examined P2X4 receptor expression in the adult mouse brain. Immunohistochemical analysis revealed that CD31-positive vascular endothelial cells expressed P2X4 receptors in the adult mouse brain. In contrast, P2X4 receptors were expressed at very low levels in NeuN-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes, and CD11b-positive microglia (Fig. 2a). Further quantitative analysis revealed that 93.3 ± 6.7% of CD31-positive cells expressed the P2X4 receptor. In other types of cells, P2X4 receptor expression was detected in 40.3 ± 15.7% of NeuN-positive cells, 10.8 ± 5.8% of GFAP-positive cells, and 15 ± 7.6% of CD11b-positive cells (Fig. 2b). To examine the necessity of P2X4 receptor expression in vascular endothelial cells for IPC-mediated neuroprotection, we generated a conditional knockout mouse in which the P2X4 receptor was knocked down in VE-cadherin-positive vascular endothelial cells. Immunohistochemical analysis confirmed that a decrease in tamoxifen-inducible Cre-mediated recombination decreased P2X4 receptor protein expression in the CD31-positive brain cells of VE-cadherin-Cre/−:: P2X4 receptor flox/flox mice compared with control mice (−/−:: P2X4 receptor flox/flox mice) (Fig. 2c). We then conducted IPC in conditional knockout mice and subjected them to prolonged MCAO. Histological analysis revealed that the infarct area was smaller in the brains of conditional knockout mice than in control mice after MCAO in the striatum and cortex (Fig. 2d–f). In addition, VE-cadherin-Cre/−:: P2X4 receptor flox/flox mice showed severe neurological deficits compared with control mice (−/−:: P2X4 receptor flox/flox mice) (Fig. 2g). These data indicate that the P2X4 receptor is required for IPC-mediated neuroprotection and the prevention of neurological damage after MCAO.
The P2X4 receptor mediates osteopontin expression in vascular endothelial cells. We next investigated the mechanism whereby P2X4 receptors expressed by vascular endothelial cells exert neuroprotective effects via IPC. Vascular endothelial cells are known to secrete factors that support cell survival26. Therefore, we hypothesized that IPC would stimulate P2X4 receptor–expressing vascular endothelial cells to enhance the Scientific Reports | 6:25893 | DOI: 10.1038/srep25893
2
www.nature.com/scientificreports/
Figure 1. P2X4 receptor inhibition abolishes neuroprotection via ischemic preconditioning. (a) Experimental design of the study. IPC (15 min MCAO) was conducted 48 hr before prolonged MCAO (60 min ischemia). 5BDBD or saline was intracerebroventricularly administered 15 min before IPC. Brain sections were obtained 24 hr after prolonged MCAO. (b) Representative images of TTC-stained brain slices. Bar, 5 mm. (c,d) Graphs show the percentage of infarction (infarct ratio) in the ipsilateral striatum (c) and cortex (d). Insertion means just insert filament into the internal carotid artery which was immediately withdrawn to make sham mice. IPC provided effective neuroprotection against brain injury caused by prolonged MCAO (striatum p = 0.0022, cortex p = 0.0008, **p
OPEN
received: 15 October 2015 accepted: 25 April 2016 Published: 13 May 2016
The P2X4 receptor is required for neuroprotection via ischemic preconditioning Tomohiko Ozaki1,2,3, Rieko Muramatsu1,2,4, Miwa Sasai5, Masahiro Yamamoto5, Yoshiaki Kubota6, Toshiyuki Fujinaka3, Toshiki Yoshimine3 & Toshihide Yamashita1,2 Ischemic preconditioning (IPC), a procedure consisting of transient ischemia and subsequent reperfusion, provides ischemic tolerance against prolonged ischemia in the brain. Although the blood flow changes mediated by IPC are primarily perceived by vascular endothelial cells, the role of these cells in ischemic tolerance has not been fully clarified. In this study, we found that the P2X4 receptor, which is abundantly expressed in vascular endothelial cells, is required for ischemic tolerance following middle artery occlusion (MCAO) in mice. Mechanistically, the P2X4 receptor was stimulated by fluid shear stress, which mimics reperfusion, thus promoting the increased expression of osteopontin, a neuroprotective molecule. Furthermore, we found that the intracerebroventricular administration of osteopontin was sufficient to exert a neuroprotective effect mediated by preconditioning-stimulated P2X4 receptor activation. These results demonstrate a novel mechanism whereby vascular endothelial cells are involved in ischemic tolerance. Ischemic brain injuries resulting from decreases in cerebral blood flow (CBF) are important causes of disability and death. Pharmacological interventions are either ineffective or confounded by adverse effects, and neuroprotection in patients after ischemic brain damage consequently remains a major unfulfilled medical need. Ischemic preconditioning (IPC), defined as transient ischemia and subsequent reperfusion, exerts neuroprotective effects against lethal ischemia. Ischemic tolerance due to preconditioning is observed in experimental animal models. In humans, the number of investigations is currently limited, but ischemic tolerance may naturally occur in human patients1. Therefore, the mechanisms of IPC have attracted considerable attention in terms of their potential applications in adaptive medicine for the treatment of ischemia. Regarding the mechanisms underlying the acquisition of ischemic tolerance, most research has focused on the involvement of neurons and astrocytes. For example, the N-methyl-D-aspartate receptor and the adenosine A1 receptor, both of which are abundantly expressed in neurons2,3, are involved in preconditioning-induced ischemic tolerance4,5. The inhibition of astrocyte-expressed P2X7 receptors attenuates the induction of ischemic tolerance6. In contrast, although IPC is caused by transient blood flow changes1 that are directly perceived by vascular endothelial cells7, the role of these cells in ischemic tolerance has not been clarified. Vascular endothelial cells are exposed to shear stress, in the form of the frictional force exerted by flowing blood. Shear stress mediates the molecular expression in vascular endothelial cells8 and influences vascular phenomena, such as angiogenesis9, atherogenesis10, and related endothelial functions11. These vascular phenomena are well known to partially regulate perivascular cells12; therefore, shear stress-mediated changes in endothelial cell molecular expression may consequently regulate perivascular cell function. Endothelial cell–derived factors, such as endothelin and prostacyclin, reportedly act on perivascular cells, regulating vascular contractility and permeability13,14. Furthermore, vascular endothelial cell–derived molecules can regulate neuronal network 1 Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 2Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. 3Department of Neurosurgery, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 4Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. 5 Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. 6The Laboratory of Vascular Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Correspondence and requests for materials should be addressed to R.M. (email: [email protected]) or T.Y. (email: [email protected])
Scientific Reports | 6:25893 | DOI: 10.1038/srep25893
1
www.nature.com/scientificreports/ formation during both development and repair processes15,16. Based on these findings, we hypothesized that endothelial cell–derived molecules, whose production is increased in response to blood flow changes, such as those involved in IPC, may benefit neurons of the central nervous system (CNS) by providing neuroprotective effects against prolonged ischemia. In this study, we focused on the role of the P2X4 receptor, a subtype of the P2X family of ligand-gated ion channels activated by adenosine triphosphate (ATP). The binding of ATP to the P2X receptor results in the entry of extracellular Ca2+ and activates intracellular signaling that evoke a variety of cellular responses. P2X4 is abundantly expressed in vascular endothelial cells17,18. P2X4−/− mice show weak responses to blood flow changes, such as low Ca2+ influx and subsequent NO production17, indicating that the P2X4 receptor is a mechanoreceptor that senses blood flow-mediated shear stress. Findings on the role of the P2X4 receptor in vascular pathology have been limited to peripheral vascular diseases, such as heart failure19. However, the P2X4 receptor has been detected in the mammalian brain20, but its function in the CNS vasculature remains unknown. Osteopontin, a secreted acidic glycoprotein containing the RGD motif, has multifunctional properties, including adhesive, chemotactic, and cytokine-like properties21. Although osteopontin is expressed in the normal brain21, the level of osteopontin increases under pathological conditions22,23. Therefore, the function of osteopontin has been studied in the context of disease. In terms of ischemic brain injury, osteopontin-deficient mice reportedly show increased neurodegeneration after ischemic cortical stroke24. Moreover, osteopontin administration prevents infarct formation after stroke in mice25. These findings led us to hypothesize that osteopontin is also involved in the endogenous neuroprotective effect mediated by IPC against ischemia, but evidence on this involvement is lacking. Here, we show that the P2X4 receptor, which is known as a shear stress sensor17, is involved in IPC-mediated neuroprotection following middle cerebral artery occlusion (MCAO)-induced cerebral ischemia in mice. P2X4 receptors are expressed by endothelial cells in the adult mouse brain. Treatment with a P2X4 receptor antagonist abolished the ability of IPC to prevent infarct formation and neurological impairment after prolonged ischemia. Shear stress also stimulated P2X4 receptors on vascular endothelial cells, leading to the upregulated expression of osteopontin and a neuroprotective effect25. Thus, neuroprotection by P2X receptor-mediated IPC might involve osteopontin up-regulation.
Results
P2X4 receptor inhibition abolishes IPC–mediated neuroprotection. We first established the experimental design (Fig. 1a) and found that the damage obtained 24 hr after 1 hr of MCAO was prevented when 15 min of MCAO (IPC) was administered 2 day before the prolonged MCAO procedure. We then examined the involvement of the P2X4 receptor in the prevention of infarct formation by IPC before prolonged MCAO. Twenty-four hours after 1 hr of MCAO, mice that received an intracerebroventricular injection of 5-BDBD, a P2X4 receptor antagonist, showed larger infarctions in both the striatum and the cortex than controls (Fig. 1b–d). IPC prevents the progression of neurological deficits caused by MCAO, and we consequently also assessed the involvement of the P2X4 receptor in this process. Mice that received a combination of 5-BDBD pretreatment and IPC showed severe neurological deficits compared with those that did not receive 5BDBD treatment (Fig. 1e). We observed that single-IPC did not form an infarct in the brain (Fig. 1b–d) or cause neurological impairment (Fig. 1e). In addition, 5-BDBD treatment did not exacerbate the infarct formation (Fig. 1b–d) or neurological impairment (Fig. 1e) caused by MCAO. Staining with 2,3,5-triphenyltetrazolium chloride (TTC) remained abundant in the brain and no neurological impairment in the intact mice (Fig. 1b–e). These data indicate that P2X4 receptor inhibition abolished IPC–mediated neuroprotection and the prevention of neurological impairment after MCAO. P2X4 receptor in vascular endothelial cells is required for neuroprotection against prolonged MCAO. To investigate the cell type(s) that are involved in P2X4 receptor-mediated neuroprotec-
tion, we examined P2X4 receptor expression in the adult mouse brain. Immunohistochemical analysis revealed that CD31-positive vascular endothelial cells expressed P2X4 receptors in the adult mouse brain. In contrast, P2X4 receptors were expressed at very low levels in NeuN-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes, and CD11b-positive microglia (Fig. 2a). Further quantitative analysis revealed that 93.3 ± 6.7% of CD31-positive cells expressed the P2X4 receptor. In other types of cells, P2X4 receptor expression was detected in 40.3 ± 15.7% of NeuN-positive cells, 10.8 ± 5.8% of GFAP-positive cells, and 15 ± 7.6% of CD11b-positive cells (Fig. 2b). To examine the necessity of P2X4 receptor expression in vascular endothelial cells for IPC-mediated neuroprotection, we generated a conditional knockout mouse in which the P2X4 receptor was knocked down in VE-cadherin-positive vascular endothelial cells. Immunohistochemical analysis confirmed that a decrease in tamoxifen-inducible Cre-mediated recombination decreased P2X4 receptor protein expression in the CD31-positive brain cells of VE-cadherin-Cre/−:: P2X4 receptor flox/flox mice compared with control mice (−/−:: P2X4 receptor flox/flox mice) (Fig. 2c). We then conducted IPC in conditional knockout mice and subjected them to prolonged MCAO. Histological analysis revealed that the infarct area was smaller in the brains of conditional knockout mice than in control mice after MCAO in the striatum and cortex (Fig. 2d–f). In addition, VE-cadherin-Cre/−:: P2X4 receptor flox/flox mice showed severe neurological deficits compared with control mice (−/−:: P2X4 receptor flox/flox mice) (Fig. 2g). These data indicate that the P2X4 receptor is required for IPC-mediated neuroprotection and the prevention of neurological damage after MCAO.
The P2X4 receptor mediates osteopontin expression in vascular endothelial cells. We next investigated the mechanism whereby P2X4 receptors expressed by vascular endothelial cells exert neuroprotective effects via IPC. Vascular endothelial cells are known to secrete factors that support cell survival26. Therefore, we hypothesized that IPC would stimulate P2X4 receptor–expressing vascular endothelial cells to enhance the Scientific Reports | 6:25893 | DOI: 10.1038/srep25893
2
www.nature.com/scientificreports/
Figure 1. P2X4 receptor inhibition abolishes neuroprotection via ischemic preconditioning. (a) Experimental design of the study. IPC (15 min MCAO) was conducted 48 hr before prolonged MCAO (60 min ischemia). 5BDBD or saline was intracerebroventricularly administered 15 min before IPC. Brain sections were obtained 24 hr after prolonged MCAO. (b) Representative images of TTC-stained brain slices. Bar, 5 mm. (c,d) Graphs show the percentage of infarction (infarct ratio) in the ipsilateral striatum (c) and cortex (d). Insertion means just insert filament into the internal carotid artery which was immediately withdrawn to make sham mice. IPC provided effective neuroprotection against brain injury caused by prolonged MCAO (striatum p = 0.0022, cortex p = 0.0008, **p
